Abstract

This research proposal is a combined biochemical and clinical investigation on the fidelity of DNA synthesis. The overall objective is to determine the mechanism in animal cells that guarantees the accurate copying of the nucleotide sequence of DNA during each division cycle. We will also investigate depurination as a common mechanism for both spontaneous and induced mutagenesis by certain carcinogens. Our specific objectives are to: 1) Identify factors in animal cells that increase the fidelity by which purified DNA polymerase copies DNA templates; 2) Isolate and analyze mutations in the gene(s) coding for DNA polymerase-alpha; 3) Determine the mutation spectrum of metal mutagenesis; 4) Measure mutagenesis as a function of apurinic sites in animal cells; 5) Investigate the relationships between tumor progression and the fidelity by which cellular DNA sequences are replicated. To accomplich these objectives, we have designed a set of assays to measure the fidelity of DNA synthesis by purified DNA polymerases and crude cellular extracts and to examine the frequency of somatic mutations in normal and malignant human cells.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Unknown (R35)
Project #
2R35CA039903-08
Application #
3479233
Study Section
Special Emphasis Panel (SRC (88))
Project Start
1985-07-15
Project End
1999-04-30
Budget Start
1992-08-01
Budget End
1993-04-30
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195

  Publications

Smith, Robert A; Loeb, Lawrence A; Preston, Bradley D (2005) Lethal mutagenesis of HIV. Virus Res 107:215-28
Shinkai, A; Patel, P H; Loeb, L A (2001) The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable. J Biol Chem 276:18836-42
Skandalis, A; Loeb, L A (2001) Enzymatic properties of rat DNA polymerase beta mutants obtained by randomized mutagenesis. Nucleic Acids Res 29:2418-26
Patel, P H; Kawate, H; Adman, E et al. (2001) A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity. J Biol Chem 276:5044-51
Patel, P H; Loeb, L A (2000) Multiple amino acid substitutions allow DNA polymerases to synthesize RNA. J Biol Chem 275:40266-72
Loeb, K R; Loeb, L A (2000) Significance of multiple mutations in cancer. Carcinogenesis 21:379-85
Patel, P H; Loeb, L A (2000) DNA polymerase active site is highly mutable: evolutionary consequences. Proc Natl Acad Sci U S A 97:5095-100
Silber, J R; Blank, A; Bobola, M S et al. (1999) O6-methylguanine-DNA methyltransferase-deficient phenotype in human gliomas: frequency and time to tumor progression after alkylating agent-based chemotherapy. Clin Cancer Res 5:807-14
Loeb, L A; Essigmann, J M; Kazazi, F et al. (1999) Lethal mutagenesis of HIV with mutagenic nucleoside analogs. Proc Natl Acad Sci U S A 96:1492-7
Encell, L P; Coates, M M; Loeb, L A (1998) Engineering human DNA alkyltransferases for gene therapy using random sequence mutagenesis. Cancer Res 58:1013-20

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